Inactivated vaccines represent a critical component of the health care system for both human and veterinary fields of medicine. However, the process of inactivation (e.g., inactivation by formaldehyde, β-propiolactone (BPL), binary ethylenimine (BEI) inactivation, and hydrogen peroxide (H2O2)) can damage key antigenic epitopes of target pathogens, leading to suboptimal in vitro and in vitro responses in vaccines and reductions in in vivo vaccine efficacy. For example, formaldehyde is an extremely reactive chemical agent and acts by forming a chemical bond with the primary amide and the amino groups of protein molecules. Thus, in vitro, it reacts with proteins, DNA and RNA, and can penetrate, e.g., even the thick wall of spores. Formaldehyde also has mutagenic potential, and its action on carboxyl, sulphydryl and hydroxyl groups makes it a strong alkylating agent. Formaldehyde forms protein-DNA cross linkages. β-propiolactone (BPL) is an alkylating agent that reacts with many nucleophilic reagents including nucleic acids and proteins. BPL modifies the structure of nucleic acids after reaction mainly with purine residues (notably guanine), induces nicks in DNA, and cross-links between DNA and proteins as well as between the DNA strands in the double helix. Consequently, BPL is widely used for the inactivation of viruses (DNA and RNA viruses). Ethyleneimine monomer (EI) or binary ethyleneimine (BEI) are used to modify (alkylate) nucleic acids preferentially at N-7, N-3, and N-1 of purines and to a lesser extent N-3 of pyrimidines. Alkylating agents enhance the opening of an imidazole ring of N-7 alkylated purines (e.g., guanine), thereby arresting replication. EI alkylates guanosine to form N-7 (aminoethyl)guanosine, which has a higher imidazole ring-opening rate than does N-7 (alkylguanosine). EI also modifies non-genomic components of viral or nonviral biomolecules. Ethyleneimine (EI) is an electrophilic inactivating agent.
Recent work (see, e.g., U.S. Pat. Nos. 8,124,397 and 8,716,000) has shown that chemical oxidizing agents (e.g., hydrogen peroxide (H2O2)), while previously known and used in the art only for the ability to destroy and kill pathogens, could be used in methods to prepare immunogenic inactivated viral vaccines. However, even such simple chemical oxidizing agents can give suboptimal results by damaging, to some extent, key antigenic epitopes, and to circumvent this problem, there is yet a pronounced unmet need for better, broadly applicable methods for inactivating pathogens while optimally retaining immunogenicity.
Influenza, for example, commonly known as “the flu”, is an infectious disease caused by an influenza virus, RNA viruses that make up three of the five genera of the family Orthomyxoviridae. Influenza spreads around the world in a yearly outbreak, resulting in about three to five million cases of severe illness and about 250,000 to 500,000 deaths.
Dengue virus (DENV), for example, is the cause of dengue fever. It is a mosquito-borne, positive-sense single stranded RNA virus of the family Flaviviridae; genus Flavivirus. Five serotypes of the virus have been found, all of which can cause the full spectrum of disease. Its genome codes for three structural proteins (capsid protein C, membrane protein M, envelope protein E) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). It also includes short non-coding regions on both the 5′ and 3′ ends.
Chikungunya virus (CHIKV), for example, is a member of the alphavirus genus, and Togaviridae family. It is an RNA virus with a positive-sense single-stranded genome of about 11.6 kb. It is a member of the Semliki Forest virus complex and is closely related to Ross River virus, O'nyong'nyong virus, and Semliki Forest virus. Because it is transmitted by arthropods, namely mosquitoes, it can also be referred to as an arbovirus (arthropod-borne virus). In the United States, it is classified as a category C priority pathogen, and work requires biosafety level III precautions. Symptoms include fever and joint pain, typically occurring two to twelve days after exposure. Other symptoms may include headache, muscle pain, joint swelling, and a rash. Most people are better within a week; however, occasionally the joint pain may last for months. The risk of death is around 1 in 1,000. The very young, old, and those with other health problems are at risk of more severe disease.
Campylobacter (Gram-negative bacteria), for example, represents a global human pathogen and is responsible for up to 400-500 million cases of bacterial gastroenteritis each year. The economic burden of this bacterial disease is substantial, with annual US costs estimated at up to $5.6 billion. There is no commercial vaccine available for human Campylobacter infections and development of a safe and effective vaccine represents an important unmet clinical need. The most frequently reported species in human diseases are C. jejuni (subspecies jejuni) and C. coli. Other species such as C. lari and C. upsaliensis have also been isolated from patients with diarrhoeal disease, but are reported less frequently.
Listeria (e.g., Listeria monocytogenes; Gram-positive bacteria) is one of the most virulent foodborne pathogens, with fatality rates due to food-borne listeriosis reaching 20 to 30% in high-risk individuals. Responsible for an estimated 1,600 illnesses and 260 deaths in the United States (U.S.) annually, listeriosis ranks third in total number of deaths among food borne bacterial pathogens, with fatality rates exceeding even Salmonella and Clostridium botulinum. In the European Union, rates of listeriosis have followed an upward trend that began in 2008, causing 2,161 confirmed cases and 210 reported deaths in 2014, 16% more than in 2013. Similar to the U.S., listeriosis mortality rates are also higher in the EU compared to other food-borne pathogens.
Shigella (e.g., Shigella dysenteriae; Gram-negative bacteria) is one of the leading bacterial causes of diarrhea worldwide, causing an estimated 80-165 million cases annually. The number of deaths it causes each year is estimated at between 74,000 and 600,000, and it is in the top four pathogens that cause moderate-to-severe diarrhea in African and South Asian children. S. flexneri is the most frequently isolated species worldwide, and accounts for 60% of cases in the developing world; S. sonnei causes 77% of cases in the developed world, compared to only 15% of cases in the developing world; and S. dysenteriae is usually the cause of epidemics of dysentery, particularly in confined populations such as refugee camps.
The present disclosure satisfies these and other needs for better vaccines.